Bispecific anti-Erb-B antibodies and their use in tumor therapy

ABSTRACT

The invention relates to novel bispecific antibodies and their use in tumor therapy. The novel antibodies have the ability to bind to ErbB receptors, preferably ErbB1 receptors, which are overexpressed on many cancer tissues. Since the different specificities of the antigen-binding sites are directed to different epitopes within the binding domain of same or different ErbB receptors, these antibodies are more effective with respect to inhibition and down-regulation of the ErbB receptor and the corresponding signaling cascade.

This application is the national phase under 35 U.S.C. 371 ofPCT/EP03/1165 filed Oct. 9, 2003.

FIELD OF THE INVENTION

The invention relates to novel bispecific antibodies and their use intumor therapy. The novel antibodies have the ability to bind to ErbBreceptors, especially ErbB1 receptors, which are overexpressed on manycancer tissues. Since the different specificities of the antigen-bindingsites are directed to different epitopes within the binding domain ofsame or different ErbB receptors, these antibodies are more effectivewith respect to inhibition and down-regulation of the ErbB receptor andthe corresponding signaling cascade. The invention relates also topharmaceutical compositions comprising said bispecific antibodies orfragments thereof and additional pharmaceutically effective agents suchas monospecific antibodies, immunoconjugates and/or cytotoxic agents.

BACKGROUND OF THE INVENTION

Biological molecules, such as monoclonal antibodies (MAbs) or otherproteins/polypeptides, as well as small chemical compounds directedagainst various receptors and other antigens on the surface of tumorcells are known to be suitable for tumor therapy for more than twentyyears. With respect to the antibody approach, most of these MAbs arechimerized or humanized to improve tolerability with the human immunesystem. Mabs or above-mentioned chemical entities specifically bind totheir target structures on tumor cells and in most cases also on normaltissues and can cause different effects that dependent on their epitopespecificity and/or functional characteristics of the particular antigen.MAbs to orphan receptors or other non-functional cell surface moleculesas well as MAbs against structures outside the ligand-binding site offunctionally active receptors (e.g. growth factor receptors with kinaseactivity) would be expected to induce primarily immune effectorfunctions against the target cell (antibody-dependent cell-mediatedcytotoxicity (ADCC), complement-dependent cytotoxicity (CDC)).Additionally, depending on the properties of antigen and MAb, binding ofthe antibody can result in cross-linking of the receptors. Consequentinternalization of the receptor-antibody complexes may result in aprolonged down-modulation of the receptor density on the cell surface.

MAbs which bind to an epitope within the ligand-binding site or in itsdirect neighborhood compete for binding of natural ligands to theirreceptor and thus reduce or completely inhibit ligand binding and candisplace already bound ligands from their receptors. This receptorblockade inhibits ligand-dependent receptor activation and downstreamsignaling. For example, blockade of ErbB receptors, such as theepidermal growth factor receptor (EGFR), by monoclonal antibodiesresults in various cellular effects including inhibition of DNAsynthesis and proliferation, induction of cell cycle arrest andapoptosis as well as antimetastatic and antiangiogenetic effects.

ErbB receptors are typical receptor tyrosine kinases that wereimplicated in cancer in the 1980s. Tyrosine kinases are a class ofenzymes that catalyze the transfer of the terminal phosphate ofadenosine triphosphate to tyrosine residues in protein substrates.Tyrosine kinases are believed, by way of substrate phosphorylation, toplay critical roles in signal transduction for a number of cellfunctions. Though the exact mechanisms of signal transduction is stillunclear, tyrosine kinases have been shown to be important contributingfactors in cell proliferation, carcinogenesis and cell differentiation.Tyrosine kinases can be categorized as receptor type or non-receptortype. Both receptor-type and non-receptor type tyrosine kinases areimplicated in cellular signaling pathways leading to numerous pathogenicconditions, including cancer, psoriasis and hyperimmune responses. Manytyrosine kinases are involved in cell growth as well as in angiogenesis.The non-receptor type of tyrosine kinases is also comprised of numeroussubfamilies, including Src, Frk, Btk, Csk, Abl, Zap70, Fes/Fps, Fak,Jak, Ack, and LIMK. Each of these subfamilies is further sub-dividedinto varying receptors. For example, the Src subfamily is one of thelargest and includes Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr, and Yrk.The Src subfamily of enzymes has been linked to oncogenesis. For a moredetailed discussion of the non-receptor type of tyrosine kinases, seeBolen Oncogene, 8:2025–2031 (1993).

Receptor type tyrosine kinases have an extracellular, a transmembrane,and an intracellular portion, while non-receptor type tyrosine kinasesare wholly intracellular. Receptor-linked tyrosine kinases aretransmembrane proteins that contain an extracellular ligand bindingdomain, a transmembrane sequence, and a cytoplasmic tyrosine kinasedomain. The receptor-type tyrosine kinases are comprised of a largenumber of transmembrane receptors with diverse biological activity.

Different subfamilies of receptor-type tyrosine kinases have beenidentified. Implicated tyrosine kinases include fibroblast growth factor(FGF) receptors, epidermal growth factor (EGF) receptors of the ErbBmajor class family, and platelet-derived growth factor (PDGF) receptors.Also implicated are nerve growth Factor (NGF) receptors, brain-derivedneurotrophic Factor (BDNF) receptors, and neurotrophin-3 (NT-3)receptors, and neurotrophin-4 (NT-4) receptors.

One receptor type tyrosine kinase subfamily, designated as HER or ErbBsubfamily, is comprised of EGFR (ErbB1), HER2 (ErbB2 or p185neu), HER3(ErbB3), and HER4(ErbB4 or tyro2). Ligands of this subfamily ofreceptors include epithelial growth factor (EGF), TGF-a, amphiregulin,HB-EGF, betacellulin, heregulin and neuregulins. The PDGF subfamilyincludes the FLK family which is comprised of the kinase insert domainreceptor (KDR).

EGFR, encoded by the erbB1 gene, has been causally implicated in humanmalignancy. In particular, increased expression of EGFR has beenobserved in breast, bladder, lung, head, neck and stomach cancer as wellas glioblastomas. Increased EGFR receptor expression is often associatedwith increased production of the EGFR ligand, transforming growth factoralpha (TGF-a), by the same tumor cells resulting in receptor activationby an autocrine stimulatory pathway (Baselga and Mendelsohn, Pharmac.Ther. 64:127–154 (1994)).

The EGF receptor is a transmembrane glycoprotein which has a molecularweight of 170.000, and is found on many epithelial cell types. It isactivated by at least three ligands, EGF, TGF-α (transforming growthfactor alpha) and amphiregulin. Both epidermal growth factor (EGF) andtransforming growth factor-alpha TGF-a) have been demonstrated to bindto EGF receptor and to lead to cellular proliferation and tumor growth.These growth factors do not bind to HER2 (Ulrich and Schlesinger, 1990,Cell 61, 203). In contrary to several families of growth factors, whichinduce receptor dimerization by virtue of their dimeric nature (e.g.PDGF) monomeric growth factors, such as EGF, contain two binding sitesfor their receptors and, therefore, can cross-link two neighboring EGFreceptors (Lemmon et al., 1997, EMBO J. 16, 281). Receptor dimerizationis essential for stimulating of the intrinsic catalytic activity and forthe self-phosphorylation of growth factor receptors on tyrosineresidues. The latter serve as docking sites for various adaptor proteinsor enzymes, which simultaneously initiate many signaling cascades. Inhigher eukaryotes, the simple linear pathway has evolved into a richlyinteractive, multi-layered network in which combinatorial expression andactivation of components permits context-specific biological responsesthroughout development. The ErbB network might integrate not only itsown inputs but also heterologous signals, including hormones,lymphokines, neurotransmitters and stress inducers.

It should be remarked that receptor protein tyrosine kinases (PTKs) areable to undergo both homo- and heterodimerization, wherein homodimericreceptor combinations are less mitogenic and transforming (no or weakinitiation of signaling) than the corresponding heterodimericcombinations. Heterodimers containing ErbB2 are the most potentcomplexes (see review articles by Yarden and Sliwkowski, 2001, NatureReviews, Molecular cell Biology, volume 2, 127–137; Tzahar and Yarden,1998, BBA 1377, M25–M37).

It has been demonstrated that anti-EGF receptor antibodies whileblocking EGF and TGF-a binding to the receptor appear to inhibit tumorcell proliferation. In view of these findings, a number of murine andrat monoclonal antibodies against EGF receptor have been developed andtested for their ability inhibit the growth of tumor cells in vitro andin vivo (Modjtahedi and Dean, 1994, J. Oncology 4, 277). Humanizedmonoclonal antibody 425 (h MAb 425, U.S. Pat. No. 5,558,864; EP 0531472) and chimeric monoclonal antibody 225 (c MAb 225, U.S. Pat. No.4,943,533 and EP 0359 282), both directed to the EGF receptor, haveshown their efficacy in clinical trials. The C225 antibody (Cetuximab)was demonstrated to inhibit EGF-mediated tumor cell growth in vitro andinhibit human tumor formation in vivo in nude mice. The antibody,moreover, appeared to act, above all, in synergy with certainchemotherapeutic agents (i.e., doxorubicin, adriamycin, taxol, andcisplatin) to eradicate human tumors in vivo in xenograft mouse models.Ye et al. (1999, Oncogene 18, 731) have reported that human ovariancancer cells can be treated successfully with a combination of bothchimeric MAb 225 and humanized MAb 4D5 which is directed to the HER2receptor.

The second member of the ErbB family, HER2 (ErbB2 or p185neu), wasoriginally identified as the product of the transforming gene fromneuroblastomas of chemically treated rats. The activated form of the neuproto-oncogene results from a point mutation (valine to glutamic acid)in the transmembrane region of the encoded protein. Amplification of thehuman homologue of neu is observed in breast and ovarian cancers andcorrelates with a poor prognosis (Slamon et al., Science, 235: 177–182(1987); Slamon et al., Science, 244:707–712 (1989); U.S. Pat. No.4,968,603). ErbB2 (HER2) has a molecular weight of about 185.000, withconsiderable homology to the EGF receptor (HER1), although a specificligand for HER2 has not yet been clearly identified so far. The antibody4D5 directed to the HER2 receptor, was further found to sensitizeErbB2-overexpressing breast tumor cell lines to the cytotoxic effects ofTNFα (U.S. Pat. No. 5,677,171). A recombinant humanized version of themurine anti-ErbB2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2 or HERCEPTIN® ;U.S. Pat. No. 5,821,337) is clinically active in patients withErbB2-overexpressing metastatic breast cancers that have receivedextensive prior anti-cancer therapy (Baselga et al., J. Clin. Oncol.14:737–744 (1996)); HERCEPTIN® received marketing approval in 1998 forthe treatment of patients with metastatic breast cancer whose tumorsoverexpress the ErbB2 protein.

Besides anti-ErbB antibodies there are numerous small chemical moleculeswhich are known to be potent inhibitors of ErbB receptor moleculesblocking the binding site of the natural ligands (see detaileddescription), or blocking the tyrosine residues of the binding site ofthe receptor kinase, thus preventing phosphorylation and further cascadesignaling. One representative showing high efficacy in clinical trialsis Iressa™ (ZD-1839) which can be applied for NSCLC indication(non-small cell lung cancer).

Although there are already some promising drugs and methods of treatmenttumors under development and in the market, there is a continuous needfor further agents and pharmaceutical compositions and combinations withimproved properties and enhanced efficacy.

SUMMARY OF THE INVENTION

The invention is based on the observation of the inventors, that certainreceptor tyrosine kinases such as ErbB receptor molecules which areoverexpressed on diseased cell surfaces, e.g. tumor cells, have specificepitope sites within the natural ligand binding domain to whichsimultaneously different antibodies or, generally spoken, differentspecificities, may be bound without or only negligible mutual hindrance.Evidently, these antibodies or specificities possess binding epitopeswhich are with respect to their three-dimensional configurationrelatively small, as compared with the total size of the binding domainof the receptor molecule. They induce an increased down-modulationactivity of pathway signaling, preferably an increased blocking of theErbB receptor and, thus, of the complete signaling cascade.

The present invention describes for the first time the new concept intumor therapy to administer to an individual a bispecific antibody or afunctionally effective fragment thereof that blocks or inhibits an ErbBreceptor, preferably the EGF receptor (EGFR), by binding of the firstspecific antigen-binding site of said bispecific antibody to a firstepitope and the second specific antigen-binding site to a seconddifferent epitope of the same or different receptor.

It could be found that such a bispecific antibody can bindsimultaneously by its two different antigen-binding sites to differentepitopes within the natural ligand(s) binding domain either of the samereceptor molecule (e.g. EGFR or Her-2) or different receptor molecules(e.g. EGFR and Her-2) without significant mutual hindrance of thedifferent antigen-binding sites of the antibody, thus enabling a higherantibody density on the receptor and affecting (by a less ability tobind natural (agonistic) ligands such as EGF or TGF a) a much strongerinhibition of the signaling cascade of the corresponding receptormolecules as monomeric or dimeric units. This should lead to a strongerinhibition of tumor growth and/or increased apoptosis of solid tumors ortumor metastases. Preferred antibodies are especially anti-EGFR andanti-Her2 antibodies as specified above and below, and fragmentsthereof, preferably bispecific F(ab′)2 fragments for reasons of theirsmaller size. In a preferred embodiment of this invention a bispecificantibody (fragment) consisting of a first antigen-binding site derivingfrom MAb 425 in a humanized, chimeric or murine version and a secondantigen-binding site deriving from MAb 225 in a humanized, chimeric ormurine version is disclosed (BAb <425, 225>, F(ab′<425>, ab′<225>)). Inanother embodiment of this invention a bispecific antibody (fragment)consisting of a first antigen-binding site deriving from MAb 425 in ahumanized, chimeric or murine version and a second antigen-binding sitederiving from MAb 4D5 in a humanized, chimeric or murine version isdisclosed (BAb <425, 4D5>, F(ab′<425>, ab′<4D5>is disclosed. In afurther embodiment of this invention a bispecific antibody (fragment)consisting of a first antigen-binding site deriving from MAb 225 in ahumanized, chimeric or murine version and a second antigen-binding sitederiving from MAb 4D5 in a humanized, chimeric or murine version isdisclosed (BAb <225, 4D5>, F(ab′<425>, ab′<4D5>)) is disclosed. Inabove-said embodiments humanized MAb 425, chimeric MAb225 (CETUXIMAB®)and humanized 4D5 (HERCEPTIN®) are preferred as source antibodies. Inprinciple the invention includes also heteroantibodies or fragmentsthereof. Such a synthetically manufactured heteroantibody may evenconsist of three different antigen-binding site portions deriving fromthree different anti-EGFR antibodies or fragments thereof (e.g. <425,225, 4D5>).

It was found that the bispecific antibodies according to this inventioncan affect enhanced cross-linking/dimerization of different or identicalErbB receptors, enhanced blocking/inhibition of ErbB receptors, andenhanced induction of modulation of ErbB receptor-specific pathwaysignaling as compared with the respective monospecific antibodies.Interestingly this cross-linking effect can be further enhanced by amixture comprising a bispecific antibody (fragment) as described and amonospecific anti-ErbB antibody (fragment), preferably havingantigen-binding sites which are identical with said first or secondantigen-binding sites of the bispecific antibosy (fragment). In otherwords: a mixture of, for example, (i) MAb 425 or MAb 225 or MAb 4D5 andBAb <425, 225>, or (ii) MAb 425 or MAb 225 or MAb 4D5 and BAb <425,4D5>, or (iii) MAb 425 or MAb 225 or MAb 4D5 and BAb <4D5, 225> elicitan enhanced inhibition and down-regulation of ErbB receptors as MAbs orBAbs applied as single agent in the same concentration.

Although above-described observations were made for ErbB receptors astarget receptor molecules only it should be pointed out that thescientific principle discovered by the inventors and stated out aboveand below might be also applicable for other biological receptors otherthan ErbB.

Optionally, the composition according to this invention comprisesfurther therapeutically active compounds which may support and enhancethe efficacy of above-said molecules. Such agents may cytotoxic agentsand preferably antagonistic molecules, such as tyrosine kinaseantagonists, other ErbB antagonists, hormone receptor antagonists,protein kinase antagonists or anti-angiogenic agents. Such moleculesusable in the present invention are specified in more detail below.

According to this invention the therapeutically active agents may alsobe provided by means of a pharmaceutical kit comprising a packagescontaining one or more of said antagonistic agents in single or separatecontainers. The therapy with this combinations may include optionallytreatment with radiation. Principally, the administration can beaccompanied by radiation therapy, wherein radiation treatment can bedone substantially concurrently or before or after the drugadministration. The administration of the different agents of thecombination therapy according to the invention can also be achievedsubstantially concurrently or sequentially. Tumors, bearing receptors ontheir cell surfaces involved in the development of the blood vessels ofthe tumor, may be successfully treated by the combination therapy ofthis invention.

It is known that tumors elicit alternative routes for their developmentand growth. If one route is blocked they often have the capability toswitch to another route by expressing and using other receptors andsignaling pathways. Therefore, the pharmaceutical combinations of thepresent invention may block several of such possible developmentstrategies of the tumor and provide consequently various benefits. Thecombinations according to the present invention are useful in treatingand preventing tumors, tumor-like and neoplasia disorders and tumormetastases, which develop and grow by activation of their relevanthormone receptors which are present on the surface of the tumor cells.Preferably, the different combined agents of the present invention areadministered in combination at a low dose, that is, at a dose lower thanhas been conventionally used in clinical situations. A benefit oflowering the dose of the compounds, compositions, agents and therapiesof the present invention administered to an individual includes adecrease in the incidence of adverse effects associated with higherdosages. For example, by the lowering the dosage of an agent describedabove and below, a reduction in the frequency and the severity of nauseaand vomiting will result when compared to that observed at higherdosages. By lowering the incidence of adverse effects, an improvement inthe quality of life of a cancer patient is contemplated. Furtherbenefits of lowering the incidence of adverse effects include animprovement in patient compliance, a reduction in the number ofhospitalizations needed for the treatment of adverse effects, and areduction in the administration of analgesic agents needed to treat painassociated with the adverse effects. Alternatively, the methods andcombination of the present invention can also maximize the therapeuticeffect at higher doses.

The combinations mentioned above show an astonishing synergetic effect.In administering the combination of drugs real tumor shrinking anddisintegration could be observed during clinical studies while nosignificant adverse drug reactions were detectable.

The invention generally refers to:

-   -   A bispecific antibody, or a functionally effective fragment        thereof, comprising a first antigen-binding site that binds to a        first epitope of a first ErbB receptor and a second different        antigen-binding site that binds to a second epitope of a second        ErbB receptor.    -   A corresponding bispecific antibody or fragment thereof, wherein        said first and/or said second epitope is located within the        binding domain of the natural ligand of said receptor(s).    -   A corresponding bispecific antibody or fragment thereof        affecting enhanced blocking and/or inhibition of ErbB receptor,        and enhanced induction of down-regulation of ErbB        receptor-specific pathway signaling as compared with the        respective monospecific antibody.    -   A corresponding bispecific antibody or fragment thereof        affecting enhanced induction of crosslinking and/or dimerization        of receptor molecules having the same or different specificity.    -   A corresponding bispecific antibody or fragment thereof, wherein        said first epitope of the first ErbB receptor is different from        the second epitope of the second ErbB receptor.    -   A bispecific antibody or fragment thereof of claim 5, wherein        said first ErbB receptor is different from said second ErbB        receptor.    -   A corresponding bispecific antibody or fragment thereof, wherein        said first and second ErbB receptors are identical.    -   A corresponding bispecific antibody or fragment thereof, wherein        said first ErbB receptor is EGF receptor (EGFR).    -   A corresponding bispecific antibody or fragment thereof, wherein        said second ErbB receptor is ErbB-2 (Her-2).    -   A corresponding bispecific antibody or fragment thereof, wherein        said first and second ErbB receptor is EGF receptor (EGFR).    -   A corresponding bispecific antibody or fragment thereof, wherein        said first antigen-binding site derives from humanized, chimeric        or murine MAb 425.    -   A corresponding bispecific antibody or fragment thereof, wherein        said first antigen-binding site derives from humanized, chimeric        or murine MAb 225.    -   A corresponding bispecific antibody or fragment thereof, wherein        said first antigen-binding site derives from humanized, chimeric        or murine MAb 425, and said second antigen-binding site derives        from humanized, chimeric or murine MAb 225, and each        antigen-binding site binds to a different epitope within the        binding domain of the natural ligand(s) of the same EGF receptor        molecule.    -   A corresponding bispecific antibody or fragment thereof, wherein        said first ErbB receptor is EGF receptor (EGFR) and said second        ErbB receptor is ErbB-2 (Her-2).    -   A corresponding bispecific antibody or fragment thereof, wherein        said first antigen-binding site derives from humanized, chimeric        or murine MAbs 425 or 225, and said second antigen-binding site        derives from MAb 4D5 (Herceptin®).    -   A corresponding bispecific antibody or fragment thereof, wherein        the fragment is F(ab′)2.    -   A pharmaceutical composition comprising a bispecific antibody or        a functionally effective fragment thereof as specified in any of        the above-mentioned claims and optionally a pharmaceutically        acceptable carrier, diluent or excipient.    -   A corresponding pharmaceutical composition further comprising a        monospecific anti-ErbB antibody or a functionally effective        fragment thereof    -   A corresponding pharmaceutical composition, wherein said        monospecific anti-ErbB antibody or a functionally effective        fragment thereof is selected from the group consisting of MAb        425, MAb 225, or MAb 4D5 (Herceptin®).    -   A corresponding pharmaceutical composition, additionally        comprising a cytotoxic agent.    -   A corresponding pharmaceutical composition, wherein said        cytotoxic agent is a chemotherapeutic agent.    -   A corresponding pharmaceutical composition, wherein said        chemotherapeutic agent is selected from any of the compounds of        the group: cisplatin, doxorubicin, gemcitabine, docetaxel,        paclitaxel, bleomycin.    -   A corresponding pharmaceutical composition, wherein said        cytotoxic agent is an ErbB receptor inhibitor, a tyrosine kinase        inhibitor, a protein kinase A inhibitor, or an anti-angiogenic        agent.    -   A pharmaceutical kit comprising        -   (i) a first package comprising at least a bispecific            antibody or a functionally effective fragment thereof as            specified above, and        -   (ii) a second package comprising at least a monospecific            anti-ErbB antibody or a functionally effective fragment            thereof.    -   A corresponding pharmaceutical kit comprising a first package        that comprises BAb <h425, c225> or its F(ab′)2 fragment, and a        second package that comprises humanized MAb 425 (h425), chimeric        MAb 225 (c225) or humanized MAb 4D5 or functionally effective        fragments thereof.    -   A corresponding pharmaceutical kit comprising additionally a        third package comprising a further agent.    -   A corresponding pharmaceutical kit, wherein said additional        agent is a cytotoxic drug.    -   A corresponding pharmaceutical kit, wherein said cytotoxic drug        is selected from any of the compounds of the group: cisplatin,        doxorubicin, gemcitabine, docetaxel, paclitaxel, bleomycin, ErbB        receptor inhibitor, a tyrosine kinase inhibitor, a protein        kinase A inhibitor, or an anti-angiogenic agent.    -   Use of a bispecific antibody or a pharmaceutical composition/kit        as defined above, for the manufacture of a medicament for the        treatment of tumors and tumor metastases that overexpress ErbB        receptors.    -   A method for treating tumors and tumor metastases that        overexpress ErbB receptors in an individual comprising        administering to said individual a therapeutically effective        amount of a bispecific antibody or functionally effective        fragment thereof or a pharmaceutical composition/kit as defined        above and in the claims.    -   A method for enhancing down-regulation of ErbB receptor-specific        pathway signaling in tumors that overexpress ErbB receptors by        administering to an individual a therapeutically effective        amount of a bispecific antibody or functionally effective        fragment thereof or a pharmaceutical composition/kit as defined        above.    -   A corresponding method, further comprising administering to the        patient an effective amount of a cytotoxic drug.    -   A corresponding method, wherein said cytotoxic agent is a        chemotherapeutic agent and is selected from any of the compounds        of the group: cisplatin, doxorubicin, gemcitabine, docetaxel,        paclitaxel, bleomycin.    -   A corresponding method, wherein said cytotoxic drug is an ErbB        receptor inhibitor, a tyrosine kinase inhibitor, a protein        kinase A inhibitor, or an anti-angiogenic agent.

In a preferred embodiment the first ErbB receptor type to which oneantigen binding site of the bispecific antibodies of the presentinvention binds is the ErbB1 receptor (EGFR). Thus, the inventionrelates in more detail to the following:

-   A bispecific antibody, or a fragment thereof, having the capability    to bind to different epitopes located on same or different ErbB    receptor molecule types, said antibody comprising a first    antigen-binding site that binds to an epitope of a first receptor    type, which is ErbB1, and a second different antigen-binding site    that binds to a different epitope of a second ErbB receptor molecule    type.-   A bispecific antibody, wherein said second ErbB receptor molecule    type is ErbB1 (EGFR).-   A bispecific antibody, wherein said second ErbB receptor molecule    type is ErbB2 (Her-2).-   A bispecific antibody, wherein at least one of said epitopes is    located within the receptor binding domain.-   A bispecific antibody, wherein said receptor binding domain is the    binding domain of the natural ligand of said receptor.-   A bispecific antibody, wherein the first or second antigen binding    site binds to an epitope within the binding domain of the natural    ligand(s) of said ErbB receptor molecule types.-   A bispecific antibody, wherein the first and second antigen binding    site binds to an epitope within the binding domain of the natural    ligand(s) of said ErbB receptor molecule types.-   A bispecific antibody, wherein the antigen binding sites bind to    different epitopes which are located on the same ErbB receptor    molecule type.-   A bispecific antibody, wherein the antigen binding sites bind to    different epitopes which are located on different ErbB receptor    molecule types.-   A bispecific antibody, wherein the first and second antigen binding    site binds each to a different epitope within the binding domain of    the natural ligand of said ErbB receptor molecule type, thus    blocking and/or inhibiting the receptor, whereby blocking and/or    inhibition of the ErbB receptor, and induction of down-regulation of    ErbB receptor-specific pathway signaling is enhanced as compared    with the respective monospecific antibody.-   A bispecific antibody, wherein induction of crosslinking and/or    dimerization of different ErbB receptor molecules having the same or    different specificity, is enhanced as compared with binding of the    bispecific antibody to epitopes on the same ErbB receptor molecule    type.-   A bispecific antibody, wherein said first antigen-binding site    derives from humanized, chimeric or murine MAb 425.-   A bispecific antibody according to any of the claims 1–11, wherein    said first antigen-binding site derives from humanized, chimeric or    murine MAb 225.-   A bispecific antibody designated as “BAb <h425, c225>”, wherein said    first antigen-binding site derives from humanized, chimeric or    murine MAb 425, and said second antigen-binding site derives from    humanized, chimeric or murine MAb 225, and each antigen-binding site    binds to a different epitope on the ErbB1 receptor (EGFR) molecule.-   A bispecific antibody, wherein said different epitopes are located    within the binding domain of the natural ligand(s).-   A bispecific antibody, wherein the second antigen binding site binds    to a ErbB2 receptor molecule (Her-2) or a VEGF receptor molecule.-   A bispecific antibody of claim 16, wherein said second    antigen-binding site derives from MAb 4D5 (Herceptin®).-   A bispecific antibody fragment deriving from a bispecific antibody    as defined above and in any of the claims, wherein the fragment is    F(ab′)2.-   A pharmaceutical composition comprising one or more of the    bispecific antibodies or fragments thereof as specified above and in    the claims, optionally together with a pharmaceutically acceptable    carrier, diluent or excipient.-   A pharmaceutical composition, further comprising a monospecific    anti-ErbB antibody or a functionally effective fragment thereof.-   A pharmaceutical composition, wherein said monospecific anti-ErbB    antibody or a functionally effective fragment thereof is selected    from the group consisting of MAb 425, MAb 225, or MAb 4D5    (Herceptin®).-   A pharmaceutical composition, additionally comprising a cytotoxic    agent.-   A pharmaceutical composition, wherein said cytotoxic agent is a    chemotherapeutic agent.-   A pharmaceutical composition, wherein said chemotherapeutic agent is    selected from any of the compounds of the group: cisplatin,    doxorubicin, gemcitabine, docetaxel, paclitaxel, bleomycin.-   A pharmaceutical composition, wherein said cytotoxic agent is an    ErbB receptor inhibitor, a VEGF receptor inhibitor, a tyrosine    kinase inhibitor, a protein kinase A inhibitor, an anti-angiogenic    agent, an anti-hormonal agent, or a cytokine.-   An immunoconjugate comprising a bispecific antibody as defined    above, fused directly or via a linker molecule via its C-terminus to    a biologically effective protein, polypeptide or peptide, wherein    preferably said protein or poly peptide is a cytokine.-   A pharmaceutical kit comprising (i) a first package comprising at    least a bispecific antibody or an immunoconjugate, as specified    above and in the claims, and (ii) a second package comprising at    least a monospecific anti-ErbB antibody or a functionally effective    fragment thereof.-   A pharmaceutical kit comprising a first package that comprises    bispecific antibody “BAb <h425, c225>” or its F(ab′)2 fragment or a    immunoconjugate thereof, and a second package comprising humanized    MAb 425 (h425), chimeric MAb 225 (c225) or humanized MAb 4D5 or    functionally effective antibody fragments or immunoconjugates    thereof.-   A pharmaceutical kit comprising additionally a third package    comprising a cytotoxic drug.-   A pharmaceutical kit, wherein said cytotoxic drug is selected from    any of the compounds of the group: cisplatin, doxorubicin,    gemcitabine, docetaxel, paclitaxel, bleomycin, an ErbB receptor    inhibitor, a VEGF receptor inhibitor, a tyrosine kinase inhibitor, a    protein kinase A inhibitor, an anti-hormonal agent, or an    anti-angiogenic agent.-   Use of a bispecific antibody or a pharmaceutical composition/kit as    defined above, for the manufacture of a medicament for the treatment    of tumors and tumor metastases and related diseases that overexpress    ErbB receptors.

DETAILED DESCRIPTION OF THE INVENTION

This invention is based on the observation that two or more MAbs withspecificities for different immunogenic structures can bind at the sametime and without or with only insignificant hindrance to their epitopes,which may be located on the same receptor and even within the samereceptor domain, e.g. within the ligand-binding domain. Therefore,bispecific antibodies with specificities for different epitopes of thesame receptor can bind to both of their specific epitopes and thus formbivalent receptor-antibody complexes, which are not seen in most caseswith monospecific MAbs on a single receptor. Alternatively, theantigen-binding sites of such bispecific antibodies can react with otheridentical receptors in near neighborhood and thus build complexesbetween these receptors. Additionally, bispecific antibodies directedagainst antigenic structures on different receptors of the same ordifferent receptor families can be used to form complexes between thesereceptors.

Application of one or more bispecific antibodies or combinations ofmono- and bispecific antibodies directed against the same or differentreceptors can greatly improve the therapeutic efficacy compared to theefficacy of treatment with only one monospecific antibody:

-   -   Each antigen-binding site of a bispecific antibody independently        binds to its specific epitopes on the target receptor (e.g.        EGFR).    -   Both antigen-binding sites of the bispecific antibody react with        their specific epitopes, which can be located on the same        receptor and possibly within the same receptor domain or on        another receptor.    -   Bispecific antibodies independently can bind to two different        epitopes on the same receptor. This increases the overall        avidity of the bispecific antibody to a single receptor compared        to the avidity of monospecific antibodies, which in most cases        is restricted to monovalent binding to that receptor.    -   Due to the higher avidity to a single receptor, compared to a        monospecific MAb, lower concentrations of the bispecific        antibody are necessary to efficiently block the receptor.    -   Because bispecific antibodies are more efficient than        monospecific MAbs for receptor blockade, they induce a more        pronounced inhibition of receptor activation and downstream        signaling.    -   Similarly, one or more bispecific antibodies or mixtures of        mono- and bispecific antibodies with specificities for different        epitopes within or near the ligand-binding domain increase the        efficacy of the receptor blockade.    -   Because receptor blockade by combinations of two or more mono-        and/or bispecific antibodies against the same receptor domain is        more effective than receptor blockade by only one single mono-        or bispecific antibody, a more effective inhibition of        ligand-binding is attained, which results in a more effective        inactivation of the receptor.    -   This more efficient receptor inactivation results in a more        effective inhibition of downstream receptor signaling and        consequently in an increased impact on ligand-dependent cell        functions.    -   Due to the more efficient receptor blockade the dosage (or        concentration) of each of the applied mono- and/or bispecific        antibodies can be reduced without loss of efficacy. This can be        of great interest when therapeutic antibodies are applied, which        show dose-limiting toxicities or side effects already below the        optimal therapeutic dose.    -   Bispecific antibodies as well as monospecific antibodies that        bind to different receptors on the same cell will first form        dimeric receptor-antibody complexes. However, due to their        different antigenic specificity, bispecific antibodies can form        receptor-antibody complexes, which are not limited to dimers of        identical receptors. Thus, receptor aggregates formed by        bispecific antibodies can contain large, theoretically unlimited        numbers of receptors.    -   These large receptor-antibody complexes improve internalization        of the receptors and thus may be more efficient for removal of        receptors from the cell surface and consequent down-modulation        of receptor-dependent cellular functions.    -   Formation of large receptor-antibody complexes can further be        enhanced by combinations of two or more mono- and/or bispecific        antibodies and thus can further enhance internalization of the        receptors and down-modulation of receptor-dependent cellular        functions.    -   Use of bispecific antibodies and combinations of two or more        mono- and/or bispecific antibodies against the same or different        receptors can be used for treatment of tumors carrying        appropriate receptors. EGFR positive tumors are a typical        example, however application of the therapeutic principle        described in this invention is not limited to this indication.        Thus, a wide variety of tumors carrying other receptors,        receptor families or other antigenic structures can be treated        using the same principle.    -   The treatment with bispecific antibodies or the combined        treatment with two or more mono- and/or bispecific antibodies        directed against different antigens on the same or different        receptors is also applicable as combination therapy together        with chemotherapeutic drugs and/or irradiation.    -   The treatment with bispecific antibodies or the combined        treatment with two or more mono- and/or bispecific antibodies        directed against different antigens on the same or different        receptors can as well be used in combination with other        therapeutic principles including but not limited to treatment        with hormone antagonists or hormone agonists, angiogenesis        inhibitors and other treatments.

The principle of combined treatment with bispecific antibodies orcombinations of mono- and bispecific antibodies with specificities todifferent antigen structures on the same or different receptors isdescribed here exemplarily for treatment of EGFR positive tumors.However, this principle is not limited to the EGFR and can be adaptedfor use with any other target antigen.

If not otherwise pointed out the terms and phrases used in thisinvention have the meanings and definitions as given below. Moreover,these definitions and meanings describe the invention in more detail,preferred embodiments included.

A “receptor” or “receptor molecule” is a soluble or membranebound/associated protein or glycoprotein comprising one or more domainsto which a ligand binds to form a receptor-ligand complex. By bindingthe ligand, which may be an agonist or an antagonist the receptor isactivated or inactivated and may initiate or block pathway signaling.

The term “receptor molecule type” or “ErbB (ErbB1) receptor moleculetype” means a specific receptor type such as ErbB1, ErbB2, etc. but nota specific single molecule of this receptor type. In other word: abispecific antibody according to the invention can bind by its firstantigen-binding site to a first epitope of an individual ErbB1 receptormolecule, whereas the second antigen binding site of this antibody bindsto a second different epitope of the same individual ErbB1 receptormolecule. It is also possible that the second antigen binding site ofthis antibody binds to a second different epitope of another individualreceptor molecule of the same type (ErbB1). Furthermore it is possiblethat the second antigen binding site of this antibody binds to a seconddifferent epitope of another individual receptor molecule of a differentErbB receptor molecule type (e.g. ErbB2).

By “ligand” or “receptor ligand” is meant a natural or syntheticcompound which binds a receptor molecule to form a receptor-ligandcomplex. The term ligand includes agonists, antagonists, and compoundswith partial agonist/antagonist action.

An “agonist” or “receptor agonist” is a natural or synthetic compoundwhich binds the receptor to form a receptor-agonist complex byactivating said receptor and receptor-agonist complex, respectively,initiating a pathway signaling and further biological processes.

By “antagonist” or “receptor antagonist” is meant a natural or syntheticcompound that has a biological effect opposite to that of an agonist. Anantagonist binds the receptor and blocks the action of a receptoragonist by competing with the agonist for receptor. An antagonist isdefined by its ability to block the actions of an agonist. A receptorantagonist may be also an antibody or an immunotherapeutically effectivefragment thereof. Preferred antagonists according to the presentinvention are cited and discussed below.

An “ErbB receptor” is a receptor protein tyrosine kinase which belongs,as already specified above, to the ErbB receptor family and includesEGFR (ErbB1), ErbB2, ErbB3 and ErbB4 receptors and other members of thisfamily to be identified in the future. The ErbB receptor will generallycomprise an extracellular domain, which may bind an ErbB ligand; alipophilic transmembrane domain; a conserved intracellular tyrosinekinase domain; and a carboxyl-terminal signaling domain harboringseveral tyrosine residues which can be phosphorylated. The ErbB receptormay be a “native sequence” ErbB receptor or an “amino acid sequencevariant” thereof. Preferably the ErbB receptor is native sequence humanErbB receptor. ErbB1 refers to the gene encoding the EGFR proteinproduct. Mostly preferred is the EGF receptor (EGFR, HER1). Theexpressions “ErbB1” and “HER1” and “EGFR” are used interchangeablyherein and refer to human HER1 protein. The expressions “ErbB2” and“HER2” are used interchangeably herein and refer to human HER2 protein.ErbB1 receptors (EGFR) are preferred according to this invention

“ErbB ligand” is a polypeptide which binds to and/or activates an ErbBreceptor. ErbB ligands which bind EGFR include, for example, EGF,TGF-alpha, amphiregulin, betacellulin, HB-EGF and epiregulin, preferablyEGF and TGF-alpha.

“ErbB receptor binding domain” is in the context of this invention thelocal region (binding sequence/loop/pocket) of the ErbB receptor towhich a natural ligand or an antagonistic or agonistic drug binds. Thisregion may comprise not only one specific binding site or epitope buttwo or more epitopes and binding sites, respectively. One specificbinding epitope within the domain binds to one kind of antagonistic oragonistic drug or ligand. Nevertheless it is deemed, that the binding ofdifferent agents to different epitopes within or nearly adjacent thebinding domain of the same receptor type generally causes by inhibitionor activation a distinct and unique signaling pathway that is typicalfor said receptor. Moreover, it should be pointed out that the phrase“within the binding domain” used in this description and claims includesalso locations in close vicinity of the real binding domain of therespective natural ligand(s).

“ErbB binding epitope or binding site” means a conformation and/orconfiguration of amino acids within or in close vicinity of the bindingdomain of an ErbB receptor to which ligands or receptorantagonists/agonists bind.

“Same ErbB/ErbB1 receptor molecule” means not necessarily the identicalreceptor molecule, but includes also another receptor molecule of thesame type. Preferably, the identical receptor molecule is meant.

The term “ErbB receptor antagonist/inhibitor” refers to a biologicallyeffective molecule, which binds and blocks or inhibits the ErbBreceptor. Thus, by blocking the receptor the antagonist prevents bindingof the ErbB ligand (agonist) and activation of the agonist/ligandreceptor complex. ErbB antagonists may be directed to HER1 (ErbB1,EGFR), HER2 (ErbB2) and ErbB3 and ErbB4. Preferred antagonists of theinvention are directed to the EGF receptor (EGFR, HER1). The ErbBreceptor antagonist may be an antibody or an immunotherapeuticallyeffective fragment thereof or non-immunobiological molecules, such as apeptide, polypeptide protein. Chemical molecules are also included,however, anti-EGFR antibodies and anti-HER2 antibodies are the preferredantagonists according to the invention.

Preferred antibodies of the invention are anti-Her1 and anti-Her2antibodies, more preferably anti-Her1 antibodies. Preferred anti-Her1antibodies are MAb 425, preferably humanized MAb 425 (hMAb 425, U.S.Pat. No. 5,558,864; EP 0531 472) and chimeric MAb 225 (CETUXIMAB®). Mostpreferred is monoclonal antibody h425, which has shown in mono-drugtherapy high efficacy combined with reduced adverse and side effects.Most preferred anti-HER2 antibody is HERCEPTIN® commercialized byGenentech/Roche. Efficacious EGF receptor antagonists according to theinvention may be also natural or synthetic chemical compounds. Someexamples of preferred molecules of this category include organiccompounds, organometallic compounds, salts of organic and organometalliccompounds. Examples for chemical HER2 receptor antagonists are: styrylsubstituted heteroaryl compounds (U.S. Pat. No. 5,656,655); bis monoand/or bicyclic aryl heteroaryl, carbocyclic, and heterocarbocycliccompounds (U.S. Pat. No. 5,646,153); tricyclic pyrimidine compounds(U.S. Pat. No. 5,679,683); quinazoline derivatives having receptortyrosine kinase inhibitory activity (U.S. Pat. No. 5,616,582);heteroarylethenediyl or heteroaryl-ethenediylaryl compounds (U.S. Pat.No. 5,196,446); a compound designated as6-(2,6-dichlorophenyl)-2-(4-(2-diethyl-aminoethoxy)phenylamino)-8-methyl-8H-pyrido(2,3) -5pyrimidin-7-one (Panek, et al.,1997, J. Pharmacol. Exp. Therap. 283,1433) inhibiting EGFR, PDGFR, andFGFR families of receptors.

The term “tyrosine kinase antagonist/inhibitor” refers according to thisinvention to natural or synthetic agents that are enabled to inhibit orblock tyrosine kinases, receptor tyrosine kinases included. Thus, theterm includes per se ErbB receptor antagonists/inhibitors as definedabove. With exception of the anti-ErbB receptor antibodies mentionedabove and below, more preferable tyrosine kinase antagonist agents underthis definition are chemical compounds which have shown efficacy inmono-drug therapy for breast and prostate cancer. Suitableindolocarbazole-type tyrosine kinase inhibitors can be obtained usinginformation found in documents such as U.S. Pat. Nos. 5,516,771;5,654,427; 5,461,146; 5,650,407. U.S. Pat. Nos. 5,475,110; 5,591,855;5,594,009 and WO 96/11933 disclose pyrrolocarbazole-type tyrosine kinaseinhibitors and prostate cancer. One of the most promising anti-canceragents in this context is gefitinib (IRESSA®, Astra Zeneca), which isreported to possess outstanding therapeutic efficacy and excellenttolerability in patients with non-small cell lung cancer (NSCLC) as wellas advanced head and neck cancer.

Preferably, the dosage of the chemical tyrosine kinase inhibitors asdefined above is from 1 pg/kg to 1 g/kg of body weight per day. Morepreferably, the dosage of tyrosine kinase inhibitors is from 0.01 mg/kgto 100 mg/kg of body weight per day.

The biological molecules according to this invention are preferablyantibodies or fragments thereof or any variations of antibodies such asimmunoconjugates.

In this context, the term “antibody” or “immunoglobulin” herein is usedin the broadest sense and specifically covers intact monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.bispecific antibodies) formed from at least two intact antibodies, andantibody fragments, so long as they exhibit the desired biologicalactivity. The term generally includes heteroantibodies which arecomposed of two or more antibodies or fragments thereof of differentbinding specificity which are linked together.

Depending on the amino acid sequence of their constant regions, intactantibodies can be assigned to different “antibody (immunoglobulin)classes”. There are five major classes of intact antibodies: IgA, IgD,IgE, IgG, and IgM, and several of these may be further divided into“subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.The heavy-chain constant domains that correspond to the differentclasses of antibodies are called α, δ, ε, γ and μ respectively.Preferred major class for antibodies according to the invention is IgG,in more detail IgG1 and IgG2.

Antibodies are usually glycoproteins having a molecular weight of about150,000, composed of two identical light (L) chains and two identicalheavy (H) chains. Each light chain is linked to a heavy chain by onecovalent disulfide bond, while the number of disulfide linkages variesamong the heavy chains of different immunoglobulin isotypes. Each heavyand light chain also has regularly spaced intrachain disulfide bridges.Each heavy chain has at one end a variable domain (VH) followed by anumber of constant domains. Each light chain has a variable domain atone end (VL) and a constant domain at its other end. The constant domainof the light chain is aligned with the first constant domain of theheavy chain, and the light-chain variable domain is aligned with thevariable domain of the heavy chain. Particular amino acid residues arebelieved to form an interface between the light chain and heavy chainvariable domains. The “light chains” of antibodies from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. Methods formaking monoclonal antibodies include the hybridoma method described byKohler and Milstein (1975, Nature 256,495) and in “Monoclonal AntibodyTechnology, The Production and Characterization of Rodent and HumanHybridomas” (1985, Burdon et al., Eds, Laboratory Techniques inBiochemistry and Molecular Biology, Volume 13, Elsevier SciencePublishers, Amsterdam), or may be made by well known recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies mayalso be isolated from phage antibody libraries using the techniquesdescribed in Clackson et al., Nature, 352:624–628 (1991) and Marks etal., J. Mol. Biol., 222:58, 1-597(1991), for example.

The term “chimeric antibody” means antibodies in which a portion of theheavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, so long as they exhibit the desired biological activity(e.g.: U.S. Pat. No. 4,816,567; Morrison et al., Proc. Nat. Acad. Sci.USA, 81:6851–6855 (1984)). Methods for making chimeric and humanizedantibodies are also known in the art. For example, methods for makingchimeric antibodies include those described in patents by Boss(Celltech) and by Cabilly (Genentech) (U.S. Pat. No. 4,816,397; U.S.Pat. No. 4,816,567).

“Humanized antibodies” are forms of non-human (e.g., rodent) chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region (CDRs) of the recipient are replaced by residuesfrom a hypervariable region of a non-human species (donor antibody) suchas mouse, rat, rabbit or nonhuman primate having the desiredspecificity, affinity and capacity. In some instances, framework region(FR) residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. Methods for making humanized antibodies are described,for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech,U.S. Pat. No. 4,816,397).

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen-binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)2, Fv and Fcfragments, diabodies, linear antibodies, single-chain antibodymolecules; and multispecific antibodies formed from antibodyfragment(s). An “intact” antibody is one which comprises anantigen-binding variable region as well as a light chain constant domain(CL) and heavy chain constant domains, CH1, CH2 and CH3. Preferably, theintact antibody has one or more effector functions. Papain digestion ofantibodies produces two identical antigen-binding fragments, called“Fab” fragments, each comprising a single antigen-binding site and a CLand a CH1 region, and a residual “Fc” fragment, whose name reflects itsability to crystallize readily.

The “Fc” region of the antibodies comprises, as a rule, a CH2, CH3 andthe hinge region of an IgG1 or IgG2 antibody major class. The hingeregion is a group of about 15 amino acid residues which combine the CH1region with the CH2–CH3 region.

Pepsin treatment yields an “F(ab′)2” fragment that has twoantigen-binding sites and is still capable of cross-linking antigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions (CDRs) of each variable domain interact to definean antigen-binding site on the surface of the VH-VL dimer. Collectively,the six hypervariable regions confer antigen-binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three hypervariable regions specific for an antigen) hasthe ability to recognize and bind antigen, although at a lower affinitythan the entire binding site.

The “Fab” fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain and has oneantigen-binding site only.

“Fab′” fragments differ from Fab fragments by the addition of a fewresidues at the carboxy terminus of the heavy chain CH1 domain includingone or more cysteines from the antibody hinge region.

F(ab′)2 antibody fragments originally were produced as pairs of Fab′.fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known (see e.g. Hermanson,Bioconjugate Techniques, Academic Press, 1996; U.S. Pat. No. 4,342,566).

“Single-chain Fv” or “scFv” antibody fragments comprise the V, and V,domains of antibody, wherein these domains are present in a Singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. Single-chain FVantibodies are known, for example, from Plückthun (The Pharmacology ofMonoclonal Antibodies, Vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269–315 (1994)), WO93/16185; U.S. Pat.No. 5,571,894; U.S. Pat. No. 5,587,458; Huston et al. (1988, Proc. Natl.Acad. Sci. 85, 5879) or Skerra and Plueckthun (1988, Science 240, 1038).

The term “variable” or “FR” refers to the fact that certain portions ofthe variable domains differ extensively in sequence among antibodies andare used in the binding and specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed throughout the variable domains of antibodies. It isconcentrated in three segments called “hypervariable” regions both inthe light chain and the heavy chain variable domains. The more highlyconserved portions of variable domains are called the framework regions(FRs). The variable domains of native heavy and light chains eachcomprise four FRs (FR1–FR4), largely adopting a β-sheet configuration,connected by three hypervariable regions, which form loops connecting,and in some cases forming part of the β-sheet structure. Thehypervariable regions in each chain are held together in close proximityby the FRs and, with the hypervariable regions from the other chain,contribute to the formation of the antigen-binding site of antibodies(see Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md.(1991)). The constant domains are not involved directly in binding anantibody to an antigen, but exhibit various effector functions, such asparticipation of the antibody in antibody dependent cellularcytotoxicity (ADCC).

The term “hypervariable region” or “CDR” when used herein refers to theamino acid residues of an antibody which are responsible forantigen-binding. The hypervariable region generally comprises amino acidresidues from a “complementarity determining region” or “CDR” (e.g.residues 24–34 (L1), 50–56 (L2) and 89–97 (L3) in the light chainvariable domain and 31–35 (H1), 50–65 (H2) and 95–102 (H3) in the heavychain variable domain; and/or those residues from a “hypervariable loop”(e.g. residues 26–32 (L1), 50–52 (L2) and 91–96 (L3) in the light chainvariable domain and 26–32 (H1), 53–55 (H2) and 96–101 (H3) in the heavychain variable domain; Chothia and Lesk J. Mol. Biol. 196:901–917(1987)). “Framework Region” or “FR” residues are those variable domainresidues other than the hypervariable region residues as herein defined.

The term “monospecific” refers to antibodies according to thisinvention, wherein the two binding sites of the antibody have the samespecificity, thus, being able to bind to the same epitope on thereceptor. Preferably, according to this invention, the pharmaceuticalcompositions comprise monospecific antibodies.

“Bispecific antibodies” (BAbs) are single, divalent antibodies (orimmunotherapeutically effective fragments thereof) which have twodifferently specific antigen binding sites. According to this inventionBAbs are characterized as BAb <MAb 1, MAb 2>, wherein <MAb 1> and <MAb2> designates the antigen-binding sites deriving from MAb 1 and MAb 2.For example the first antigen binding site is directed to anangiogenesis receptor (e.g. integrin or VEGF receptor), whereas thesecond antigen binding site is directed to an ErbB receptor (e.g. EGFRor HER2). Bispecific antibodies can be produced by chemical techniques(see e.g., Kranz et al. (1981) Proc. Natl. Acad. Sci. USA 78, 5807), by“polydoma” techniques (See U.S. Pat. No. 4,474,893) or by recombinantDNA techniques, which all are known per se. Further methods aredescribed in WO 91/00360, WO 92/05793 and WO 96/04305. Bispecificantibodies can also be prepared from single chain antibodies (see e.g.,Huston et al. (1988) Proc. Natl. Acad. Sci. 85, 5879; Skerra andPlueckthun (1988) Science 240, 1038). These are analogues of antibodyvariable regions produced as a single polypeptide chain. To form thebispecific binding agent, the single chain antibodies may be coupledtogether chemically or by genetic engineering methods known in the art.It is also possible to produce bispecific antibodies according to thisinvention by using leucine zipper sequences. The sequences employed arederived from the leucine zipper regions of the transcription factors Fosand Jun (Landschulz et al., 1988, Science 240,1759; for review, seeManiatis and Abel, 1989, Nature 341, 24). Leucine zippers are specificamino acid sequences about 20–40 residues long with leucine typicallyoccurring at every seventh residue. Such zipper sequences formamphipathic α-helices, with the leucine residues lined up on thehydrophobic side for dimer formation. Peptides corresponding to theleucine zippers of the Fos and Jun proteins form heterodimerspreferentially (O'Shea et al., 1989, Science 245, 646). Zippercontaining bispecific antibodies and methods for making them are alsodisclosed in WO 92/10209 and WO 93/11162.

The term “fusion protein” refers to a natural or synthetic moleculeconsisting of one ore more biological molecules as defined above,wherein two or more peptide- or protein-based (glycoproteins included)molecules having different specificity are fused together optionally bychemical or amino acid based linker molecules. The linkage may beachieved by C—N fusion or N—C fusion (in 5′→3′ direction), preferablyC—N fusion. Preferred fusion proteins according to the invention are,however, immunoconjugates as defines below.

The term “immunoconjugate” refers to a fusion protein and means anantibody or immunoglobulin, respectively, or a immunologically effectivefragment thereof, which is fused by covalent linkage to anon-immunologically effective molecule. Preferably this fusion partneris a peptide or a protein, which may be glycosylated. Said non-antibodymolecule can be linked to the C-terminal of the constant heavy chains ofthe antibody or to the N-terminals of the variable light and/or heavychains. The fusion partners can be linked via a linker molecule, whichis, as a rule, a 3–15 amino acid residues containing peptide.Immunoconjugates according to this invention are fusion proteinsconsisting of an immunoglobulin or immunotherapeutically effectivefragment thereof, directed to an ErbB receptor, and preferably acytokine, such as TNFα, IFNγ or IL-2, or another toxic agent.Preferably, these peptide- or protein-based molecules are linked withtheir N-terminal to the C-terminal of said immunoglobulin, which is theFc portion thereof.

“Heteroantibodies” are fusion proteins consisting essentially of two ormore antibodies or antibody-binding fragments which are fused togetherby regularly chemical cross-linkers, each of said antibodies having adifferent binding specificity. Heteroantibodies can be prepared byconjugating together two or more antibodies or antibody fragments.Preferred heteroantibodies are comprised of cross-linked Fab/Fab′fragments. A variety of coupling or cross-linking agents can be used toconjugate the antibodies. Examples are protein A, carboiimide,N-succinimidyl-S-acetyl-thioacetate (SATA) andN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (see e.g.,Karpovsky et al. (1984) J. EXP. Med. 160,1686; Liu et a. (1985) Proc.Natl. Acad. Sci. USA 82, 8648). Other methods include those described byPaulus, Behring Inst. Mitt., No. 78, 118 (1985); Brennan et al. (1985)Science 30, 81, or Glennie et al. (1987), J. Immunol. 139, 2367. Anothermethod uses o-phenylenedimaleimide (oPDM) for coupling three Fab′fragments (WO 91/03493). Multispecific antibodies are in context of thisinvention also suitable and can be prepared, for example according tothe teaching of WO 94/13804 and WO 98/50431. A preferred heteroantibodyaccording to this invention is a fusion protein comprising two anti-EGFRantibodies (each antibody is directed to different epitopes of the samereceptor) linked together as described (e.g. MAB 425–MAB225).

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; mouse gonadotropin-associatedpeptide; inhibin; activin; vascular endothelial growth factor (VEGF);integrin; thrombopoietin (TPO); nerve growth factors such as NGFβ;platelet-growth factor; transforming growth factors (TGFs) such as TGFαand TGFβ; erythropoietin (EPO); interferons such as IFNα, IFNβ, andIFNγ; colony stimulating factors such as M-CSF, GM-CSF and G-CSF;interleukins such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-7, IL-8,IL-9, IL-10, IL-11,IL-12; and TNF-αor TNF-β. Preferred cytokinesaccording to the invention are interferons, TNFα and IL-2.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include complement dependent cytotoxicity, Fcreceptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC),phagocytosis; down regulation of cell surface receptors (e.g. B cellreceptor), etc.

The term “ADCC” (antibody-dependent cell-mediated cytotoxicity) refersto a cell-mediated reaction in which nonspecific cytotoxic cells thatexpress Fc receptors (FcR) (e.g. natural killer (NK) cells, neutrophils,and macrophages) recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. The primary cells formediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. To assess ADCC activity of a moleculeof interest, an in vitro ADCC assay, such as that described in the priorart (U.S. Pat. No. 5,500,362; U.S. Pat. No. 5,821,337) may be performed.Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and natural killer (NK) cells.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and FcγRIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcRs are reviewed, for example, inRavetch and Kinet, Annu. Rev. Immunol 9:457–92 (1991).

The therapeutic approach of this invention includes as a specificembodiment the administration of further therapeutically effectiveagents, which support the desired effect, e.g. tumor toxicity orcytostatic efficacy, or diminish or prevent undesired side effects. Thusthe invention includes the combination of such agents with thepharmaceutical composition defined and claimed above and below, whereinsaid agents may be other ErbB receptor antagonists, VEGF receptorantagonists, cytokines, cytokine-immunoconjugates, anti-angiogenicagents, anti-hormonal agents, or cytotoxic agents in general. It is alsoan object of this invention to combine the compositions as definedherein with radiotherapy according to known methods.

The term “cytotoxic agent” as used in this context is defined verybroadly and refers to a substance that inhibits or prevents the functionof cells and/or causes destruction of cells (cell death), and/or exertsanti-neoplastic/anti-proliferative effects, for example, preventsdirectly or indirectly the development, maturation or spread ofneoplastic tumor cells. The term includes expressively also such agentsthat cause a cytostatic effect only and not a mere cytotoxic effect. Theterm includes chemotherapeutic agents as specified below, as well asother ErbB antagonists (such as anti-ErbB antibodies), anti-angiogenicagents, tyrosine kinase inhibitors, protein kinase A inhibitors, membersof the cytokine family, radioactive isotopes, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin.

The term “chemotherapeutic agent” is a subset of the term “cytotoxicagent” and means tumor cell, and less indirectly through mechanisms suchas biological response modification. Suitable chemotherapeutic agentsaccording to the invention are preferably natural or synthetic chemicalcompounds. There are large numbers of anti-neoplastic chemical agentsavailable in commercial use, in clinical evaluation and in pre-clinicaldevelopment, which could be included in the present invention fortreatment of tumors/neoplasia by combination therapy with the receptorantagonists as claimed and described in this invention. It should bepointed out that the chemotherapeutic agents can be administeredoptionally together with said ErbB receptor antagonists, preferably saidanti-EGFR antibodies, according to the invention.

Examples of chemotherapeutic or agents include alkylating agents, forexample, nitrogen mustards, ethyleneimine compounds, alkyl sulphonatesand other compounds with an alkylating action such as nitrosoureas,cisplatin and dacarbazine; antimetabolites, for example, folic acid,purine or pyrimidine antagonists; mitotic inhibitors, for example, vincaalkaloids and derivatives of podophyllotoxin; cytotoxic antibiotics andcamptothecin derivatives.

Preferred chemotherapeutic agents are amifostine (ethyol), cisplatin,dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard),streptozocin, cyclophosphamide, carrnustine (BCNU), lomustine (CCNU),doxorubicin (adriamycin), doxorubicin lipo (doxil), gemcitabine(gemzar), daunorubicin, daunorubicin lipo (daunoxome), procarbazine,mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil (5-FU),vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel(taxotere), aldesleukin, asparaginase, busulfan, carboplatin,cladribine, camptothecin, CPT-11, 10-hydroxy-7-ethyl-camptothecin(SN38), gefitinib (Iressa), dacarbazine, floxuridine, fludarabine,hydroxyurea, ifosfamide, idarubicin, mesna, interferon alpha, interferonbeta, irinotecan, mitoxantrone, topotecan, leuprolide, megestrol,melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase,pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen,teniposide, testolactone, thioguanine, thiotepa, uracil mustard,vinorelbine, chlorambucil and combinations thereof.

Most preferred chemotherapeutic agents according to the invention arecisplatin, gemcitabine, doxorubicin, paclitaxel (taxol) and bleomycin.

An “anti-angiogenic agent” refers to a natural or synthetic compoundwhich blocks, or interferes with to some degree, the development ofblood vessels. The anti-angiogenic molecule may, for instance, be abiological molecule that binds to and blocks an angiogenic growth factoror growth factor receptor. The preferred anti-angiogenic molecule hereinbinds to an receptor, preferably to an integrin receptor or to VEGFreceptor. The term includes according to the invention also a prodrug ofsaid angiogenic agent. The term furthermore includes agents effective asdescribed and also classified as cytotoxic, preferably, chemotherapeuticagents.

There are a lot of molecules having different structure and origin whichelicit anti-agiogenic properties. Most relevant classes of angiogenesisinhibitong or blocking agents which are suitable in this invention, are,for example:

-   (i) anti-mitotics such as flurouracil, mytomycin-C, taxol;-   (ii) estrogen metabolites such as 2-methoxyestradiol;-   (iii) matrix metalloproteinase (MMP) inhibitors, which inhibit zinc    metalloproteinases metalloproteases) (e.g. betimastat, BB16, TIMPs,    minocycline, GM6001, or those described in “Inhibition of Matrix    Metalloproteinases: Therapeutic Applications” (Golub, Annals of the    New York Academy of Science, Vol. 878a; Greenwald, Zucker (Eds.),    1999);-   (iv) anti-angiogenic multi-functional agents and factors such as    IFNα (U.S. Pat. No. 4,530,901; U.S. Pat. No. 4,503,035; 5,231,176);    angiostatin and plasminogen fragments (e.g. kringle 1–4, kringle 5,    kringle 1–3 (O'Reilly, M. S. et al., Cell (Cambridge, Mass.) 79(2):    315–328, 1994; Cao et al., J. Biol. Chem. 271: 29461–29467, 1996;    Cao et al., J. Biol. Chem. 272: 22924–22928, 1997); endostatin    (O'Reilly, M. S. et al., Cell 88(2), 277, 1997 and WO 97/15666),    thrombospondin (TSP-1; Frazier, 1991, Curr Opin Cell Biol 3(5):    792); platelet factor 4 (PF4);-   (v) plasminogen activator/urokinase inhibitors;-   (vi) urokinase receptor antagonists;-   (vii) heparinases;-   (viii) fumagillin analogs such as TNP-470;-   (ix) tyrosine kinase inhibitors such as SUl 0. Many of the above and    below mentioned ErbB receptor antagonists (EGFR/HER2 antagonists)    are also tyrosine kinase inhibitors, and may show, therefore    anti-EGF receptor blocking activity which results in inhibiting    tumor growth, as well as anti-angiogenic activity which results in    inhibiting the development of blood vessels and endothelial cells,    respectively;-   (x) suramin and suramin analogs;-   (xi) angiostatic steroids;-   (xii) VEGF and bFGF antagonists;-   (xiii) VEGF receptor antagonists such as anti-VEGF receptor    antibodies (DC-101);-   (xiv) flk-1 and flt-1 antagonists;-   (xv) cyclooxxygenase-II inhibitors such as COX-II;-   (xvi) integrin antagonists and integrin receptor antagonists such as    αv antagonists and αv receptor antagonists, for example, anti-αv    receptor antibodies and RGD peptides. Integrin (receptor)    antagonists are preferred according to this invention. The term    “integrin antagonists/inhibitors” or “integrin receptor    antagonists/inhibitors” refers to a natural or synthetic molecule    that blocks and inhibit an integrin receptor. In some cases, the    term includes antagonists directed to the ligands of said integrin    receptors (such as for α_(v)β₃: vitronectin, fibrin, fibrinogen, von    Willebrand's factor, thrombospondin, laminin; for α_(v)β₅:    vitronectin; for α_(v)β₁: fibronectin and vitronectin; for α_(v)β₆:    fibronectin).    -   Antagonists directed to the integrin receptors are preferred        according to the invention. Integrin (receptor) antagonists may        be natural or synthetic peptides, non-peptides, peptidomimetica,        immunoglobulins, such as antibodies or functional fragments        thereof, or immunoconjugates (fusion proteins).    -   Preferred integrin inhibitors of the invention are directed to        receptor of α_(v) integrins (e.g. α_(v)β₃, α_(v)β₅, α_(v)β₆ and        sub-classes). Preferred integrin inhibitors ar α_(v)        antagonists, and in particular α_(v)β₃ antagonists. Preferred        α_(v) antagonists according to the invention are RGD peptides,        peptidomimetic (non-peptide) antagonists and anti-integrin        receptor antibodies such as antibodies blocking α_(v) receptors.        Exemplary, non-immunological α_(v)β₃ antagonists are described        in the teachings of U.S. Pat. No. 5,753,230 and U.S. Pat. No.        5,766,591. Preferred antagonists are linear and cyclic        RGD-containing peptides. Cyclic peptides are, as a rule, more        stable and elicit an enhanced serum half-life. The most        preferred integrin antagonist of the invention is, however,        cyclo-(Arg-Gly-Asp-DPhe-NMeVal) (EMD 121974, Cilengitide®, Merck        KgaA, Germany; EP 0770 622) which is efficacious in blocking the        integrin receptors α_(v)β₃, α_(v)β₁, α_(v)β₆, α_(v)β₈,        α_(IIb)β₃.    -   Suitable peptidic as well as peptido-mimetic (non-peptide)        antagonists of the α_(v)β₃/α_(v)β₅/α_(v)β₆ integrin receptor        have been described both in the scientific and patent        literature. For example, reference is made to Hoekstra and        Poulter, 1998, Curr. Med. Chem. 5, 195; WO 95/32710; WO        95/37655; WO 97/01540; WO 97/37655; WO 97/45137; WO 97/41844; WO        98/08840; WO 98/18460; WO 98/18461; WO 98/25892; WO 98/31359; WO        98/30542; WO 99/15506; WO 99/15507; WO 99/31061; WO 00/06169; EP        0853 084; EP 0854 140; EP 0854 145; U.S. Pat. No. 5,780,426; and        U.S. Pat. No. 6,048,861. Patents that disclose benzazepine, as        well as related benzodiazepine and benzocycloheptene α_(v)β₃        integrin receptor antagonists, which are also suitable for the        use in this invention, include WO 96/00574, WO 96/00730, WO        96106087, WO 96/26190, WO 97/24119, WO 97124122, WO 97/24124, WO        98/15278, WO 99/05107, WO 99/06049, WO 99/15170, WO 99/15178, WO        97/34865, WO 97/01540, WO 98/30542, WO 99/11626, and WO        99/15508. Other integrin receptor antagonists featuring backbone        conformational ring constraints have been described in WO        98/08840; WO 99/30709; WO 99/30713; WO 99/31099; WO 00/09503;        U.S. Pat. No. 5,919,792; U.S. Pat. No. 5,925,655; U.S. Pat. No.        5,981,546; and U.S. Pat. No. 6,017,926. In U.S. Pat. No.        6,048,861 and WO 00/72801 a series of nonanoic acid derivatives        which are potent α_(v)β₃ integrin receptor antagonists were        disclosed. Other chemical small molecule integrin antagonists        (mostly vitronectin antagonists) are described in WO 00/38665.        Other α_(v)β₃ receptor antagonists have been shown to be        effective in inhibiting angiogenesis.    -   For example, synthetic receptor antagonists such as        (S)-10,11-Dihydro-3-[3-(pyridin-2-ylamino)-1-propyloxy]-5H-dibenzo[a,d]cycloheptene-10-acetic        acid (known as SB-265123) have been tested in a variety of        mammalian model systems. (Keenan et al., 1998, Bioorg. Med.        Chem. Lett. 8(22), 3171; Ward et al., 1999, Drug Metab. Dispos.        27(11), 1232). Assays for the identification of integrin        antagonists suitable for use as an antagonist are described,        e.g. by Smith et al., 1990, J. Biol. Chem. 265, 12267, and in        the referenced patent literature. Anti-integrin receptor        antibodies are also well known. Suitable anti-integrin (e.g.        α_(v)β₃, α_(v)β₅, α_(v)β₆) monoclonal antibodies can be modified        to encompasses antigen binding fragments thereof, including        F(ab)₂, Fab, and engineered Fv or single-chain antibody. One        suitable and preferably used monoclonal antibody directed        against integrin receptor α_(v)β₃ is identified as LM609 (Brooks        et al., 1994, Cell 79, 1157; ATCC HB 9537). A potent specific        anti-α_(v)β₅ antibody, P1F6, is disclosed in WO 97/45447, which        is also preferred according to this invention. A further        suitable α_(v)β₆ selective antibody is MAb 14D9.F8 (WO 99/37683,        DSM ACC2331, Merck KGaA, Germany) as well as MAb 17.E6 (EP 0719        859, DSM ACC2160, Merck KGaA) which is selectively directed to        the α_(v)-chain of integrin receptors. Another suitable        anti-integrin antibody is the commercialized Vitraxin®.

As used herein, the term “anti-hormonal agent” includes natural orsynthetic, organic or peptidic compounds that act to regulate or inhibithormone action on tumors. In more detail an “anti-hormonal agent” (1)inhibits the production of serum androgens, (2) blocks binding of serumandrogens to androgen receptors, or (3) inhibits the conversion oftestosterone to DHT, or a combination of two or more such compounds. Ananti-hormonal agent according to the invention includes in generalsteroid receptor antagonists and in more detail anti-estrogens includingfor example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, andtoremifene (Fareston); and anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptablesalts, acids or derivatives of any of the above. The term includes alsoagonists and/or antagonists of glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH) and LHRH (leuteinizing hormone—releasinghormone). A LHRH agonist useful in this invention is goserelin acetate,commercially available as ZOLADEX© (Zeneca). Another example of a usefulLHRH antagonist is GANIRELIX© (Roche/Akzo Nobel). Examples of steroidalanti-androgens are cyproterone acetate (CPA) and megestrol acetate,commercially available as MEGACE® (Bristol-Myers Oncology). Steroidalanti-androgens may block prostatic androgen receptors. They may alsoinhibit the release of LH. CPA is preferably administered to humanpatients at dosages of 100 mg/day to 250 mg/day. Nonsteroidalanti-androgens block androgen receptors. They may also cause an increasein serum LH levels and Serum testosterone levels. A preferrednonsteroidal anti-androgen is flutamide (2-methyl-N-[4-20nitro-3-(trifluoromethyl)phenyl]propanamide), commercially available asEULEXIN® (Schering Corp.). Flutamide exerts is anti-androgenic action byinhibiting androgen uptake, by inhibiting nuclear binding of androgen intarget tissues, or both. Another non-steroidal anti-androgen isnilutamide, whose chemical name is5,5-dimethyl-3-[4-nitro-3-(trifluoromethyl-4′-nitrophenyl)-4,4-dimethyl-imidazolidine-dione.In some embodiments of the invention, the anti-hormonal agent is acombination of an LHRH agonist such as leuprolide acetate, and anantiandrogen such as flutamide or nilutamide. For example, leuprolideacetate can be administered by subcucaneous, intramuscular orintravenous injection, and concurrently the flutamide can beadministered orally. Anti-hormonal agents according to the inventioninclude, as pointed out above, antagonists of the steroid/thyroidhormone receptors, including antagonists for other non-permissivereceptors, such as antagonists for RAR, TR, VDR, and the like. Asreadily recognized by those of skill in the art, a variety of retinoicacid receptor (RAR) antagonists, both synthetic and naturally occurring,can be used in accordance with the present invention.

The bispecific antibodies according to this invention can be combinedwith other drugs. These drugs can be preferably selected as follows:

-   -   tyrosine kinase inhibitors, such as Iressa®;    -   anti-angiogenic agents, preferably integrin inhibitors, more        preferably RGD peptides, cyclic peptides included, such as        cyclo-(Arg-Gly-Asp-DPhe-NMeVal) (Cilengitide®, Merck KGaA);    -   anti-VEGF receptor antibodies, such as DC-101, or VEGF        antagonists;    -   COX-II inhibitors;    -   cytokines, such as TNF-α, IFN-α, IFN-β, IFN-γ, IL-2;    -   type I protein kinase A (PKAI) inhibitors, such as mixed        backbone antisense oligonucleotides, like HYB 165 (see, for        example, Tortora et al., 1999, Clin. Cancer Res., 875–881);    -   anti-hormonal agents, such as goserelin, boserelin, leuprorelin,        tamoxifen.

The terms “cancer” and “tumor” refer to or describe the physiologicalcondition in mammals that is typically characterized by unregulated cellgrowth. By means of the pharmaceutical compositions according of thepresent invention tumors can be treated such as tumors of the breast,heart, lung, small intestine, colon, spleen, kidney, bladder, head andneck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood,thymus, uterus, testicles, cervix, and liver. More specifically thetumor is selected from the group consisting of adenoma, angio-sarcoma,astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma,hamartoma, hemangioendothelioma, hemangiosarcoma, hematoma,hepato-blastoma, leukemia, lymphoma, medulloblastoma, melanoma,neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcomaand teratoma. In detail, the tumor is selected from the group consistingof acral lentiginous melanoma, actinic keratoses, adenocarcinoma,adenoid cycstic carcinoma, adenomas, adenosarcoma, adenosquamouscarcinoma, astrocytic tumors, bartholin gland carcinoma, basal cellcarcinoma, bronchial gland carcinomas, capillary, carcinoids, carcinoma,carcinosarcoma, cavernous, cholangio-carcinoma, chondosarcoma, choriodplexus papilloma/carcinoma, clear cell carcinoma, cystadenoma,endodermal sinus tumor, endometrial hyperplasia, endometrial stromalsarcoma, endometrioid adenocarcinoma, ependymal, epitheloid, Ewing'ssarcoma, fibrolamellar, focal nodular hyperplasia, gastrinoma, germ celltumors, glioblastoma, glucagonoma, hemangiblastomas,hemangioendothelioma, hemangiomas, hepatic adenoma, hepaticadenomatosis, hepatocellular carcinoma, insulinoma, intraepithelialneoplasia, interepithelial squamous cell neoplasia, invasive squamouscell carcinoma, large cell carcinoma, leiomyosarcoma, lentigo malignamelanomas, malignant melanoma, malignant mesothelial tumors,medulloblastoma, medulloepithelioma, melanoma, meningeal, mesothelial,metastatic carcinoma, mucoepidermoid carcinoma, neuroblastoma,neuroepithelial adenocarcinoma nodular melanoma, oat cell carcinoma,oligodendroglial, osteosarcoma, pancreatic polypeptide, papillary serousadeno-carcinoma, pineal cell, pituitary tumors, plasmacytoma,pseudo-sarcoma, pulmonary blastoma, renal cell carcinoma,retinoblastoma, rhabdomyo-sarcoma, sarcoma, serous carcinoma, small cellcarcinoma, soft tissue carcinomas, somatostatin-secreting tumor,squamous carcinoma, squamous cell carcinoma, submesothelial, superficialspreading melanoma, undifferentiated carcinoma, uveal melanoma,vermucous carcinoma, vipoma, well differentiated carcinoma, and Wilm'stumor.

Tumors which can be preferably be treated with the antibody moleculesaccording to the invention are solid tumors or tumor metastases thatexpress ErbB receptors, especially ErbB1 receptors, in high amounts,such as breast cancer, prostate cancer head and neck cancer, SCLC,pancreas cancer.

The term “biologically/functionally effective” or “therapeuticallyeffective (amount)” refers to a drug/molecule which causes a biologicalfunction or a change of a biological function in vivo or in vitro, andwhich is effective in a specific amount to treat a disease or disorderin a mammal, preferably in a human. In the case of cancer, thetherapeutically effective amount of the drug may reduce the number ofcancer cells; reduce the tumor size; inhibit (i.e., slow to some extentand preferably stop) cancer cell infiltration into peripheral organs;inhibit (i.e., slow to some extent and preferably stop) tumormetastasis; inhibit, to some extent, tumor growth; and/or relieve tosome extent one or more of the symptoms associated with the cancer. Tothe extent the drug may prevent growth and/or kill existing cancercells, it may be cytostatic and/or cytotoxic. For cancer therapy,efficacy can, for example, be measured by assessing the time to diseaseprogression (TTP) and/or determining the response rate (RR).

The term “immunotherapeutically effective” refers to biologicalmolecules which cause an immune response in a mammal. More specifically,the term refers to molecules which may recognize and bind an antigen.Typically, antibodies, antibody fragments and antibody fusion proteinscomprising their antigen binding sites (complementary determiningregions, CDRs) are immunotherapeutically effective.

“Radiotherapy”: According to the invention the tumors can additionallybe treated with radiation or radiopharmaceuticals. The source ofradiation can be either external or internal to the patient beingtreated. When the source is external to the patient, the therapy isknown as external beam radiation therapy (EBRT). When the source ofradiation is internal to the patient, the treatment is calledbrachytherapy (BT). Some typical radioactive atoms that have been usedinclude radium, cesium-137, and iridium-192, americium-241 and gold-198,Cobalt-57; Copper-67; Technetium-99; Iodide-123; Iodide-131; andIndium-111. It is also possible to label the agents according to theinvention with radioactive isotopes. Today radiation therapy is thestandard treatment to control unresectable or inoperable tumors and/ortumor metastases. Improved results have been seen when radiation therapyhas been combined with chemotherapy. Radiation therapy is based on theprinciple that high-dose radiation delivered to a target area willresult in the death of reproductive cells in both tumor and normaltissues. The radiation dosage regimen is generally defined in terms ofradiation absorbed dose (rad), time and fractionation, and must becarefully defined by the oncologist. The amount of radiation a patientreceives will depend on various consideration but the two most importantconsiderations are the location of the tumor in relation to othercritical structures or organs of the body, and the extent to which thetumor has spread. A preferred course of treatment for a patientundergoing radiation therapy will be a treatment schedule over a 5 to 6week period, with a total dose of 50 to 60 Gy administered to thepatient in a single. daily fraction of 1.8 to 2.0 Gy, 5 days a week. AGy is an abbreviation for Gray and refers to a dose of 100 rad. Iftumors are treated with the anti-ErbB antibodies as described in thisinvention in context with a radiation regimen, usually a positive andeven synergistic effect can be observed. In other words, the inhibitionof tumor growth by means of said compounds is enhanced when combinedwith radiation and/or chemotherapeutic agents. Radiation therapy can beoptionally used according to the invention. It is recommended andpreferred in cases in which no sufficient amounts of the agentsaccording to the invention can be administered to the patient.

“Pharmaceutical treatment”: The method of the invention comprises avariety of modalities for practicing the invention in terms of thesteps. For example, the agents according to the invention can beadministered simultaneously, sequentially, or separately. Furthermore,the agents can be separately administered within a time interval ofabout 3 weeks between administrations, i.e., from substantiallyimmediately after the first active agent is administered to up to about3 weeks after the first agent is administered. The method can bepracticed following a surgical procedure. Alternatively, the surgicalprocedure can be practiced during the interval between administration ofthe first active agent and the second active agent. Exemplary of thismethod is the combination of the present method with surgical tumorremoval. Treatment according to the method will typically compriseadministration of the therapeutic compositions in one or more cycles ofadministration. For example, where a simultaneous administration ispracticed, a therapeutic composition comprising both agents isadministered over a time period of from about 2 days to about 3 weeks ina single cycle. Thereafter, the treatment cycle can be repeated asneeded according to the judgment of the practicing physician. Similarly,where a sequential application is contemplated, the administration timefor each individual therapeutic will be adjusted to typically cover thesame time period. The interval between cycles can vary from about zeroto 2 months.

The agents of this invention can be administered parenterally byinjection or by gradual infusion over time. Although the tissue to betreated can typically be accessed in the body by systemic administrationand therefore most often treated by intravenous administration oftherapeutic compositions, other tissues and delivery means arecontemplated where there is a likelihood that the tissue targetedcontains the target molecule. Thus, the agents of this invention can beadministered intraocularly, intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, transdermally, byorthotopic injection and infusion, and can also be delivered byperistaltic means. The therapeutic compositions containing, for example,an integrin antagonist of this invention are conventionally administeredintravenously, as by injection of a unit dose, for example.

Therapeutic compositions of the present invention contain aphysiologically tolerable carrier together with the relevant agent asdescribed herein, dissolved or dispersed therein as an activeingredient.

As used herein, the term “pharmaceutically acceptable” refers tocompositions, carriers, diluents and reagents which represent materialsthat are capable of administration to or upon a mammal without theproduction of undesirable physiological effects such as nausea,dizziness, gastric upset and the like. The preparation of apharmacological composition that contains active ingredients dissolvedor dispersed therein is well understood in the art and need not belimited based on formulation. Typically, such compositions are preparedas injectables either as liquid solutions or suspensions, however, solidforms suitable for solution, or suspensions, in liquid prior to use canalso be prepared. The preparation can also be emulsified. The activeingredient can be mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient and in amountssuitable for use in the therapeutic methods described herein. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanolor the like and combinations thereof. In addition, if desired, thecomposition can contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents and the like whichenhance the effectiveness of the active ingredient. The therapeuticcomposition of the present invention can include pharmaceuticallyacceptable salts of the components therein. Pharmaceutically acceptablesalts include the acid addition salts (formed with the free amino groupsof the polypeptide) that are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, tartaric, mandelic and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like. Particularly preferred is theHCI salt when used in the preparation of cyclic polypeptide α_(v)antagonists. Physiologically tolerable carriers are well known in theart. Exemplary of liquid carriers are sterile aqueous solutions thatcontain no materials in addition to the active ingredients and water, orcontain a buffer such as sodium phosphate at physiological pH value,physiological saline or both, such as phosphate-buffered saline. Stillfurther, aqueous carriers can contain more than one buffer salt, as wellas salts such as sodium and potassium chlorides, dextrose, polyethyleneglycol and other solutes. Liquid compositions can also contain liquidphases in addition to and to the exclusion of water. Exemplary of suchadditional liquid phases are glycerin. vegetable oils such as cottonseedoil, and water-oil emulsions.

Typically, a therapeutically effective amount of an immunotherapeuticagent, for example, in the form of an ErbB (ErbB1) receptor blockingbispecific antibody, an integrin receptor blocking antibody or antibodyfragment or antibody conjugate or an anti-VEGF receptor blockingantibody, fragment or conjugate is an amount such that, whenadministered in physiologically tolerable composition, is sufficient toachieve a plasma concentration of from about 0.01 microgram (μg) permilliliter (ml) to about 100 μg/ml, preferably from about 1 g/ml toabout 5 μg/ml and usually about 5 μg/ml. Stated differently. the dosagecan vary from about 0.1 mg/kg to about 300 mg/kg, preferably from about0.2 mg/kg to about 200 mg/kg, most preferably from about 0.5 mg/kg toabout 20 mg/kg, in one or more dose administrations daily for one orseveral days. Where the immunotherapeutic agent is in the form of afragment of a monoclonal antibody or a conjugate, the amount can readilybe adjusted based on the mass of the fragment/conjugate relative to themass of the whole antibody. A preferred plasma concentration in molarityis from about 2 micromolar (μM) to about 5 millimolar (mM) andpreferably, about 100 μM to 1 mM antibody antagonist.

A therapeutically effective amount of an agent according of thisinvention which is a non-immunotherapeutic peptide or a proteinpolypeptide or other similarly-sized biological molecule, is typicallyan amount of polypeptide such that when administered in aphysiologically tolerable composition is sufficient to achieve a plasmaconcentration of from about 0.1 microgram (μg) per milliliter (ml) toabout 200 μg/ml, preferably from about 1 μg/ml to about 150 μg/ml. Basedon a polypeptide having a mass of about 500 grams per mole, thepreferred plasma concentration in molarity is from about 2 micromolar(μM) to about 5 millimolar (mM and preferably about 100 μM to 1 mMpolypeptide antagonist.

The typical dosage of an active agent, which is a preferably a chemicalcytotoxic or chemotherapeutic agent according to the invention (neitheran immunotherapeutic agent nor a non-immunotherapeutic peptide/protein)is 10 mg to 1000 mg, preferably about 20 to 200 mg, and more preferably50 to 100 mg per kilogram body weight per day. The pharmaceuticalcompositions of the invention can comprise phrase encompasses treatmentof a subject with agents that reduce or avoid side effects associatedwith the combination therapy of the present invention (“adjunctivetherapy”), including, but not limited to, those agents, for example,that reduce the toxic effect of anticancer drugs, e.g., bone resorptioninhibitors, cardioprotective agents. Said adjunctive agents prevent orreduce the incidence of nausea and vomiting associated withchemotherapy, radiotherapy or operation, or reduce the incidence ofinfection associated with the administration of myelosuppressiveanticancer drugs. Adjunctive agents are well known in the art. Theimmunotherapeutic agents according to the invention can additionallyadministered with adjuvants like BCG and immune system stimulators.Furthermore, the compositions may include immunotherapeutic agents orchemotherapeutic agents which contain cytotoxic effective radio-labeledisotopes, or other cytotoxic agents, such as a cytotoxic peptides (e.g.cytokines) or cytotoxic drugs and the like.

The term “pharmaceutical kit” for treating tumors or tumor metastasesrefers to a package and, as a rule, instructions for using the reagentsin methods to treat tumors and tumor metastases. A reagent in a kit ofthis invention is typically formulated as a therapeutic composition asdescribed herein, and therefore can be in any of a variety of formssuitable for distribution in a kit. Such forms can include a liquid,powder, tablet, suspension and the like formulation for providing thepharmaceutical molecules of this invention, preferably the anti-ErbB1antibodies. The reagents may be provided in separate containers suitablefor administration separately according to the present methods, oralternatively may be provided combined in a composition in a singlecontainer in the package. The package may contain an amount sufficientfor one or more dosages of reagents according to the treatment methodsdescribed herein. A kit of this invention also contains “instructionsfor use” of the materials contained in the package.

EXAMPLES Example 1 Preparation of F(ab′)2-Fragments of MAb 425 and MAb225

Anti-EGFR antibodies humanized MAb 425 and chimeric MAb 225 wereconverted into F(ab′)2-fragments by limited proteolysis. The generationof F(ab′)2 antibody had to be optimized for each antibody. A generalscheme used for the production is given below. Pepsin cleavage was bestfor both antibodies, however papain cleavage is also applicable.Residual complete antibody and Fc-fragments were removed with a ProteinA sepharose column. The yield of F(ab′)2-fragment was close to 100%. TheF(ab′)2-fragments can be stored at −20° C. without any loss of activityfor an extended time period.

General Scheme:

Growth fermentation→Centrifugation→Ultrafiltration→Protein AChromatography→Dialysis/Ultrafiltration→Cleavage→Protein AChromatography→Dialysis/Ultrafiltration→F(ab′)2 product

Details:

-   PBS, pH: 7.4-   Protein Content: 5.06 mg/ml (Pierce).    Reagents: Tri-sodium citrate dihydrate, citric acid,    tris(hydroxymethyl) aminomethane, pepsin, glycine, sodium chloride    Buffer Solutions:    -   0.1 M Na-Citrate buffer pH 3.5,    -   10 mg/mL pepsin in Na-citrate buffer pH 3.5,    -   1 M Tris pH 11    -   1.5 M glycine+3 M NaCl pH 8.9    -   0.1 M citric acid pH 2.5        Procedure for Pepsin Digestion:

pH and buffer conditions were adjusted by dialysis of MAbs over night in0.1 M sodium citrate, pH: 3.5. Pepsin was added to the dialysedimmunoglobulin in a ratio of 1:33 w/w. The mixture was continuouslystirred at 37° C. in a water bath. After 75 min the digest was stoppedby the addition of 7 ml of 1 M Tris-Solution. At this step the pH of thereaction should be set to approximately 8.5. This mixture was thentransferred to a Protein A column in order to remove residual IgG and/orFc-fragments.

Protein-A-Sepharose:

The pepsin digest was applied to an equilibrated Protein-A-Sepharosecolumn and washed with the equilibration buffer until the chromatogramreturns to the base line. The flow through fractions were collected andthe volume is reduced in an Amicon chamber (Membrane YM 30) and thendialysed against PBS pH: 7.4. Potential contaminants such asFc-fragments or unmodified antibodies were eluted from theProtein-A-Sepharose column with 0.1 M citric acid pH: 2.5.

Chromatographic Conditions:

Column bed size 5 cm×2.5 cm. 1 ml of Protein A Sepharose is expected tobind 10 mg of IgG was equilibrated with 1.5 M glycine+3 M NaCl pH: 8.9,flow-Rate: 60 ml/h, detection: OD 280 nm, 0.2/2.0 Abs-Range, Uvicord SI1, chart-Speed: 0.1 mm/min , 5 ml per fraction collected.

Yield of F(ab′)2 preparation (Pierce)

Taking into consideration that the Fc-part represents roughly one thirdof the molecule, the yield of the F(ab′)2 preparation was close to 100%for both antibodies. The concentration of the sample should be 6–7mg/ml. Purity of the F(ab′)2 preparation was monitored by SDS-PAGE.

Example 2 Preparation of Bispecific Antibody BAb <425,225>

Bispecific antibodies were generated by chemical recombination of IgGfragments as described by Brennan et al. (Science, 1985, 229, 81–83).The individual steps of the modified procedure according to theinvention are listed as follows:

-   F(ab′)2 product→Reduction to    Fab′→Gelchromatography→Derivatization→Gelchromatography→Conjugation→Gelchromatography→Ultrafiltration→Sterile    Filtration→BAbs

Both specific F(ab′)2-fragments were converted into Fab′ fragments. Thesuccess of the conjugation step was dependent on the selection of theappropriate Fab′-fragment for Ellman's modification. In the case of theMAb 225/MAb 425 derived BAb the <225> component was modified. Afterintroduction of these modifications the yield of the individual BAb's)ranged from 20–30%. Anti-225 Fab′ was modified with Ellman's reagent andconjugated to the 425 specific Fab′. Bispecific antibody was recoveredby gelfiltration chromatography.

Details:

In this step both antibodies were reduced with DTT in order to generateFab′ fragments. The MAb 225 derived Fab′ was modified with Ellman'sreagent prior to conjugation. It is however possible to modify MAb 425derived Fab′ with Ellmann's reagent.

Fragments:

-   (i) F(ab′<425>)2 7.4 mg/ml-   (ii) F(ab′<225>)2 6.9 mg/ml    Solution/Buffers:

PBS pH 7.4, PBS+0.65 M NaCl+2.5 mM EDTA, pH 7.4, 51 mM Dithiotreitol inPBS, 0.1 M Na-phosphate buffer, pH 8.0, 35 mM Ellman's reagent in 0.1 MNa-phosphate buffer, pH 8.0, 250 mM EDTA, pH7.4

Reagents:

1,4-Dithiotreitol , Ellman's-Reagent, sodium chloride , disodiumorthophosphate, potassium dihydrogen phosphate, EDTA, Titriplex III,Superdex 200 (26/60) Pharmacia,

First Step of Synthesis:

Preparation of MAb<225>Fab′-TNB.

-   6550 μl F(ab′)2 MAb 225 40 mg+65.4 μl DTT 51 mM+65.4 μl EDTA 250 mM.-   End concentration of DTT was 0.5 mM and 2.5 mM for EDTA.

The reaction was overlayed with argon and incubated in water bath at 30°C. for 40 min under continuously stirring. After incubation, 1120 μl ofEllman's reagent were added to the reaction mixture, this stepreversibly blocks the free SH-groups in the resulting Fab′. The finalconcentration of Ellman's in the reaction is 5.0 mM. The reactionmixture was stirred at RT for 30 min in order to block all theSH-groups. The colour of the reaction mixture changes from clear toyellow. The reaction mixture was purified using a Superdex 200 (26/60)column with PBS+0.65M NaCl+2.5 mM EDTA buffer, to separate the reducedEllman's modified Fab′ molecules from potential contaminantes such asunreduced F(ab′)2, Fab′ and surplus of reagent. The Fab-TNB fractionswere pooled, overlayed with argon and stored on ice until the couplingreaction.

Second Step of Synthesis:

Preparation of MAb <425> Fab′.

-   6135 μl F(ab′)2 MAb 425 40 mg+80 μl EDTA 250 mM+80 μl DTT 51 mM.-   Start The reaction should be started not before the Fab-TNB    preparation was almost finished. End concentration of reaction is    0.5 mM DTT and 2.5 mM EDTA. Reaction mixture was overlayed with    argon and incubated at 30° C. for 40 min. Immediately after    incubation, reaction mixture was transferred to the equilibrated    Superdex 200 (26/60) column, using PBS+0.65 M NaCl+2.5 mM EDTA pH    7.4 buffer to separate Fab′ from unreduced F(ab′)2 and DTT. The    buffer and collecting tube were argon saturated respectively, to    prevent oxidation of the free SH groups. Fab′ containing fractions    were collected directly in an argon-saturated test-tube.    Third Step of Synthesis:-   Conjugation of Fab′<425> and Fab′<225>-TNB

Coupling reaction: 32.5 ml MAb 225 Fab′-TNB, 0.9 mg/ml, 31.6 mg+23.5 mlMAb 425 Fab′ 1.5 mg/ml, 34.8 mg. The Fab′ and the Fab′-TNB antibodieswere combined and the volume was reduced to approx. 5 ml (using argon)in an Amicon chamber containing a YM 10 membrane. The reaction mixturewas overlayed with argon at 4° C. overnight under continuous stirring.The conjugate was purified through a Superdex 200 (26/60) column, thebuffer and column were helium-saturated. Bispecific F(ab′)2, (Peak 1)were recovered. Peak 1: purified bispecific antibody (166–187 ml), Peak2: residual Fab′. To confirm identity and purity samples were applied toa non-reducing 10% SDS-Page gel. The yield of purified BAb <425, 225>F(ab′)2 was in this typical example 11 mg (16.7%).

1. A bispecific antibody, or a fragment thereof, having the capabilityto bind to EGF receptor (EGFR), said antibody comprising a firstantigen-binding site that binds to a first epitope of said EGFR, and asecond different antigen-binding site that binds to a second epitope ofsaid EGFR, wherein said first antigen-binding site is humanized,chimeric, or murine MAb425 and said second antigen-binding site ishumanized, chimeric, or murine MAb225, and each of said first and secondantigen-binding site binds to a different epitope on the same EGFRmolecule.
 2. The bispecific antibody of claim 1, wherein said differentepitopes are located within the binding domain of the natural ligand(s)of said receptor.
 3. The bispecific antibody of claim 1, where at leastone of said epitopes is located within the binding domain of the naturalligand(s) of said EGF receptor.
 4. The bispecific antibody of claim 1,wherein the first or second antigen binding site binds to an epitopewithin the binding domain of the natural ligand(s) of said EGF receptormolecule.
 5. A bispecific antibody fragment of claim 1, wherein thefragment is F(ab′)2.
 6. An immunoconjugate comprising the bispecificantibody of claim 1, or a fragment thereof, fused directly or via alinker molecule via its C-terminus to a protein, polypeptide, orpeptide.
 7. The immunoconjugate of claim 6, wherein said protein is acytokine.
 8. A pharmaceutical composition comprising a bispecificantibody of claim 1 optionally together with a pharmaceuticallyacceptable carrier, diluent, or excipient.
 9. A pharmaceuticalcomposition comprising an immunoconjugate of claim 7 together with apharmaceutically acceptable carrier, diluent, or excipient.
 10. Thepharmaceutical composition claim 8 additionally comprising a cytotoxicagent.
 11. The pharmaceutical composition claim 9 additionallycomprising a cytotoxic agent.
 12. The pharmaceutical composition claim10, wherein said cytotoxic agent is a chemotherapeutic agent.
 13. Thepharmaceutical composition claim 11, wherein said cytotoxic agent is achemotherapeutic agent.
 14. The pharmaceutical composition claim 12,wherein said chemotherapeutic agent comprises at least one compoundselected from a group comprising cisplatin, doxorubicin, gemcitabine,docetaxel, paclitexel, and belomycin.
 15. The pharmaceutical compositionclaim 13, wherein said chemotherapeutic agent comprises at least onecompound selected from a group comprising cisplatin, doxorubicin,gemcitabine, docetaxel, paclitexel, and belomycin.
 16. Thepharmaceutical composition claim 10, wherein said cytotoxic agent is anErbB receptor inhibitor, a VEGF receptor inhibitor, a tyrosine kinaseinhibitor, a protein kinase A inhibitor, an anti-angiogenic agent, ananti-hormonal agent, or a cytokine.
 17. The pharmaceutical compositionclaim 11, wherein said cytotoxic agent is an ErbB receptor inhibitor, aVEGF receptor inhibitor, a tyrosine kinase inhibitor, a protein kinase Ainhibitor, an anti-angiogenic agent, an anti-hormonal agent, or acytokine.
 18. A bispecific antibody or an F(ab′)2 fragment thereofhaving the capability to bind to EGF receptor (EGFR), said antibodycomprising a first antigen-binding site that binds to a first epitope ofsaid EGFR, and a second different antigen-binding site that binds to asecond epitope of said EGFR, wherein said first antigen-binding sitederives from humanized, chimeric, or murine MAb425 and said secondantigen-binding site derives from humanized, chimeric, or murine MAb225,and each of said first and second antigen-binding site binds to adifferent epitope on the same EGFR molecule.